Genome-edited Plants Research Articles

A Simple Heat Treatment Increases SpCas9-Mediated Mutation Efficiency in Arabidopsis

The CRISPR/Cas9 system is now commonly employed for genome editing in various plants such as Arabidopsis, rice and tobacco. In general, in genome editing of the Arabidopsis genome, the SpCas9 and guide RNA genes are introduced into the genome by the floral dip method. Mutations induced in the target sequence by SpCas9 are confirmed after selecting transformants by screening the T1 seed population. The advantage of this method is that genome-edited plants can be isolated easily. However, mutation efficiency in Arabidopsis using SpCas9 is not as high as that achieved in rice and tobacco, which are subjected to a tissue culture step. In this study, we compared four promoters and found that the parsley UBIQITIN promoter is highly active in Arabidopsis meristem tissue. Furthermore, we examined whether a simple heat treatment could improve mutation efficiency in Arabidopsis. Just one heat treatment at 37°C for 24 h increased the mutation efficiency at all four target sites from 3 to 42%, 43 to 62%, 54 to 75% and 89 to 91%, without detectable off-target mutations. We recommend heat treatment of plate-grown plants at 37°C for 24 h as a simple method to increase the efficiency of CRISPR/Cas9-mediated mutagenesis in Arabidopsis.

Impacts of the regulatory environment for gene editing on delivering beneficial products.

Various genome-editing technologies have been embraced by plant breeders across the world as promising tools for the improvement of different crops to deliver consumer benefits, improve agronomic performance, and increase sustainability. The uptake of genome-editing technologies in plant breeding greatly depends on how governments regulate its use. Some major agricultural production countries have already developed regulatory approaches that enable the application of genome editing for crop improvement, while other governments are in the early stages of formulating policy. Central to the discussion is the principle of “like products should be treated in like ways” and the subsequent utilization of exclusions and exemptions from the scope of GMO regulations for these products. In some countries, the outcomes of genome editing that could also have been achieved through conventional breeding have been defined as not needing GMO regulatory oversight. In this paper, we provide a short overview of plant breeding and the history of plant biotechnology policy development, the different classes of current regulatory systems and their use of exemptions and exclusions for genome-edited plants, and the potential benefits of such approaches as it relates to achieving societal goals.

Targeted mutagenesis in Nicotiana tabacum ADF gene using shockwave-mediated ribonucleoprotein delivery increases osmotic stress tolerance.

DNA-free genome editing involves the direct introduction of ribonucleoprotein (RNP) complexes into cells, but this strategy has rarely been successful in plants. In the present study, we describe a new technique for the introduction of RNPs into plant cells involving the generation of cavitation bubbles using a pulsed laser. The resulting shockwave achieves the efficient transfection of walled cells in tissue explants by creating transient membrane pores. RNP-containing cells were rapidly identified by fluorescence microscopy, followed by regeneration and the screening of mutant plants by high-resolution melt analysis. We used this technique in Nicotiana tabacum to target the endogenous phytoene desaturase (PDS) and actin depolymerizing factor (ADF) genes. Genome-edited plants were produced with an efficiency of 35.2% for PDS and 16.5% for ADF. Further we evaluated the physiological, cellular and molecular effects of ADF mutations in T2 mutant plants under drought and salinity stress. The results suggest that ADF acts as a key regulator of osmotic stress tolerance in plants.

Future-Proofing EU Legislation for Genome-Edited Plants: Dutch Stakeholders’ Views on Possible Ways Forward

Genome editing is an emerging, new breeding technology with numerous potential applications in plant breeding. In Europe, genome editing is regarded, in legal terms, as a genetic modification technique, hence plants obtained using these methods fall under the legislation for genetically modified organisms (GMOs). Despite the opportunities that genome editing brings to the plant sector, it also poses challenges to the regulatory system. For example, the enforcement of labelling and traceability requirements for GM foods and feeds may be impossible for small genome edits that are indistinguishable from natural mutations. In order to discuss potential adaptations of EU legislation with stakeholders from the Dutch plant breeding sector, five different scenarios of future regulation of plants obtained by means of genome editing were elaborated. These scenarios were discussed in depth, along with the potential applications of genome editing in plant breeding, as well as challenges and opportunities. Stakeholders particularly indicated their preference for new, future-proof legislation in the long term, which will also include products of novel technologies. Finally, we discuss potential short-term amendments to current legislation, including the exemption of certain small mutations, that would make it align with regulation of genome edited plants in non-EU countries.

Economic and Environmental Consequences of the ECJ Genome Editing Judgment in Agriculture

Genome-edited crops are on the verge of being placed on the market and their agricultural and food products will thus be internationally traded soon. National regulations, however, diverge regarding the classification of genome-edited crops. Major countries such as the US and Brazil do not specifically regulate genome-edited crops, while in the European Union, they fall under GMO legislation, according to the European Court of Justice (ECJ). As it is in some cases impossible to analytically distinguish between products from genome-edited plants and those from non-genome-edited plants, EU importers may fear the risk of violating EU legislation. They may choose not to import any agricultural and food products based on crops for which genome-edited varieties are available. Therefore, crop products of which the EU is currently a net importer would become more expensive in the EU, and production would intensify. Furthermore, an intense substitution of products covered and not covered by genome editing would occur in consumption, production, and trade. We analyzed the effects of such a cease of EU imports for cereals and soy in the EU agricultural sector with the comparative static agricultural sector equilibrium model CAPRI. Our results indicate dramatic effects on agricultural and food prices as well as on farm income. The intensification of EU agriculture may result in negative net environmental effects in the EU as well as in an increase in global greenhouse gas (GHG) emissions. This suggests that trade effects should be considered when developing domestic regulation for genome-edited crops.

An Editing-Site-Specific PCR Method for Detection and Quantification of CAO1-Edited Rice.

Genome-edited plants created by genome editing technology have been approved for commercialization. Due to molecular characteristics that differ from classic genetically modified organisms (GMOs), establishing regulation-compliant analytical methods for identification and quantification of genome-edited plants has always been regarded as a challenging task. An editing-site-specific PCR method was developed based on the unique edited sequence in CAO1-edited rice plants. Test results of seven primer/probe sets indicated that this method can identify specific CAO1-edited rice from other CAO1-edited rice and wild types of rice with high specificity and sensitivity. The use of LNA (locked nucleic acid) in a probe can efficiently increase the specificity of the editing-site-specific PCR method at increased annealing temperature which can eliminate non-specific amplification of the non-target. The genome-edited ingredient content in blinded samples at the level of 0.1% to 5.0% was accurately quantified by this method on the ddPCR platform with RSD of <15% and bias in the range of ±17%, meeting the performance requirements for GMO detection method. The developed editing-site-specific PCR method presents a promising detection and quantification technique for genome-edited plants with known edited sequence.

High-Throughput and Low-Cost Genotyping Method for Plant Genome Editing.

Genome editing technologies have revolutionized genetic studies in the life sciences community in recent years. The application of these technologies allows researchers to conveniently generate mutations in almost any gene of interest. This is very useful for species such as maize that have complex genomes and lack comprehensive mutant collections. With the improvement of genome editing tools and transformation methods, these technologies are also widely used to assist breeding research and implementation in maize. However, the detection and genotyping of genomic edits rely on low-throughput, high-cost methods, such as traditional agarose gel electrophoresis and Sanger sequencing. This article describes a method to barcode the target regions of genomic edits from many individuals by low-cost polymerase chain reaction (PCR) amplification. It also employs next-generation sequencing (NGS) to genotype the genome-edited plants at high throughput and low cost. This protocol can be used for initial screening of genomic edits as well as derived population genotyping on a small or large scale, at high efficiency and low cost. © 2021 Wiley Periodicals LLC. Basic Protocol 1: A fast genomic DNA preparation method from genome edited plants Basic Protocol 2: Barcoding the amplicons of edited regions from each individual by two rounds of PCR Basic Protocol 3: Bioinformatics analysis.

Genome Editing: Revolutionizing the Crop Improvement

Modern biotechnology is progressed to sequence-specific, site-directed, precise, and safe strategies for genetic manipulation. The genome-editing strategy is being proposed as one of the most promising tools for genomic study and crop improvement. The basic mechanism involved in genetic manipulations through programmable nucleases is recognition of target genomic loci and binding of effector DNA-binding domain (DBD), double-strand breaks (DSBs) in target DNA by the restriction endonucleases (FokI and Cas) and the repair of DSBs through homology-directed recombination (HDR) or non-homologous end joining (NHEJ). The less efficient and more precise HDR results in the replacement of nucleotides, whereas the more efficient and error-prone NHEJ results in the insertion or deletion of nucleotides. The genome-editing strategies like ZFN, TALEN, and CRISPR/Cas involved in genome editing are being employed to add the desirable trait(s) and remove the undesirable. The impact of gene editing application in crop improvement has proven it the next-generation tool to enhance agricultural productivity. The modifications in crop plants include biotic and abiotic stresses, nutritional improvements, alteration in phenotype, and other characters such as crafting male sterility, haploid production, and increase in yield. The repurposing of CRISPR applications by catalytically inactive Cas9 marks CRISPR as an indispensable tool for genome editing and beyond in biological research. However, the genome-edited plants may have unintended alteration(s) that create biosafety risks. The tools are eventually progressing for more accuracy and efficiency to ensure the safety of consumers and the environment. This article is an attempt to review these genome-editing strategies, the biochemistry attributed to application in crop improvement, and the associated challenges.

Implications of the EFSA Scientific Opinion on Site Directed Nucleases 1 and 2 for Risk Assessment of Genome-Edited Plants in the EU

Genome editing is a set of techniques for introducing targeted changes in genomes. It may be achieved by enzymes collectively called site-directed nucleases (SDN). Site-specificity of SDNs is provided either by the DNA binding domain of the protein molecule itself or by RNA molecule(s) that direct SDN to a specific site in the genome. In contrast to transgenesis resulting in the insertion of exogenous DNA, genome editing only affects specific endogenous sequences. Therefore, multiple jurisdictions around the world have exempted certain types of genome-edited organisms from national biosafety regulations completely, or on a case-by-case basis. In the EU, however, the ruling of the Court of Justice on the scope of mutagenesis exemption case C-528/16 indicated that the genome-edited organisms are subject to the GMO Directive, but the practical implications for stakeholders wishing to develop and authorize genome-edited products in the EU remain unclear. European Food Safety Authority in response to a request by European Commission has produced a scientific opinion on plants developed by SDN-1, SDN-2, and oligonucleotide-directed mutagenesis (ODM) genome editing techniques. In this review, I will (1) provide a conceptual background on GMO risk assessment in the EU; (2) will introduce the main conclusions of the EFSA opinion, and (3) will outline the potential impact on the risk assessment of genome-edited plants.

In planta Genome Editing in Commercial Wheat Varieties.

Limitations for the application of genome editing technologies on elite wheat (Triticum aestivum L.) varieties are mainly due to the dependency on in vitro culture and regeneration capabilities. Recently, we developed an in planta particle bombardment (iPB) method which has increased process efficiency since no culture steps are required to create stably genome-edited wheat plants. Here, we report the application of the iPB method to commercially relevant Japanese elite wheat varieties. The biolistic delivery of gold particles coated with plasmids expressing CRISPR/Cas9 components designed to target TaQsd1 were bombarded into the embryos of imbibed seeds with their shoot apical meristem (SAM) exposed. Mutations in the target gene were subsequently analyzed within flag leaf tissue by using cleaved amplified polymorphic sequence (CAPS) analysis. A total of 9/358 (2.51%) of the bombarded plants (cv. “Haruyokoi,” spring type) carried mutant alleles in the tissue. Due to the chimeric nature of the T0 plants, only six of them were inherited to the next (T1) generation. Genotypic analysis of the T2 plants revealed a single triple-recessive homozygous mutant of the TaQsd1 gene. Compared to wild type, the homozygous mutant exhibited a 7 days delay in the time required for 50% seed germination. The iPB method was also applied to two elite winter cultivars, “Yumechikara” and “Kitanokaori,” which resulted in successful genome editing at slightly lower efficiencies as compared to “Haruyokoi.” Taken together, this report demonstrates that the in planta genome editing method through SAM bombardment can be applicable to elite wheat varieties that are otherwise reluctant to callus culture.

Genome-Edited Plants: Opportunities and Challenges for an Anticipatory Detection and Identification Framework.

It is difficult to trace and identify genome-edited food and feed products if relevant information is not made available to competent authorities. This results in major challenges, as genetically modified organism (GMO) regulatory frameworks for food and feed that apply to countries such as the member states of the European Union (EU) require enforcement based on detection. An international anticipatory detection and identification framework for voluntary collaboration and collation of disclosed information on genome-edited plants could be a valuable tool to address these challenges caused by data gaps. Scrutinizing different information sources and establishing a level of information that is sufficient to unambiguously conclude on the application of genome editing in the plant breeding process can support the identification of genome-edited products by complementing the results of analytical detection. International coordination to set up an appropriate state-of-the-art database is recommended to overcome the difficulty caused by the non-harmonized bio-safety regulation requirements of genome-edited food and feed products in various countries. This approach helps to avoid trade disruptions and to facilitate GMO/non-GMO labeling schemes. Implementation of the legal requirements for genome-edited food and feed products in the EU and elsewhere would substantially benefit from such an anticipatory framework.

The Di2/pet Variant in the PETALOSA Gene Underlies a Major Heat Requirement-Related QTL for Blooming Date in Peach [Prunus persica (L.) Batsch

Environmental adaptation of deciduous fruit trees largely depends on their ability to synchronize growth and development with seasonal climate change. Winter dormancy of flower buds is a key process to prevent frost damage and ensure reproductive success. Temperature is a crucial environmental stimulus largely influencing the timing of flowering, only occurring after fulfillment of certain temperature requirements. Nevertheless, genetic variation affecting chilling or heat-dependent dormancy release still remains largely unknown. In this study, a major QTL able to delay blooming date in peach by increasing heat requirement was finely mapped in three segregating progenies, revealing a strict association with a genetic variant (petDEL) in a PETALOSA gene, previously shown to also affect flower morphology. Analysis of segregating genome-edited tobacco plants provided further evidence of the potential ability of PET variations to delay flowering time. Potential applications of the petDEL variant for improving phenological traits in peach are discussed.

Detection Methods Fit-for-Purpose in Enforcement Control of Genetically Modified Plants Produced with Novel Genomic Techniques (NGTs)

The comprehensive EU regulatory framework regarding GMOs aims at preventing damage to human and animal health and the environment, and foresees labelling and traceability. Genome-edited plants and products fall under these EU GMO regulations, which have to be implemented in enforcement control activities. GMO detection methods currently used by enforcement laboratories are based on real-time PCR, where specificity and sensitivity are important performance parameters. Genome editing allows the targeted modification of nucleotide sequences in organisms, including plants, and often produces single nucleotide variants (SNVs), which are the most challenging class of genome edits to detect. The test method must therefore meet advanced requirements regarding specificity, which can be increased by modifying a PCR method. Digital PCR systems achieve a very high sensitivity and have advantages in quantitative measurement. Sequencing methods may also be used to detect DNA modifications caused by genome editing. Whereas most PCR methods can be carried out in an enforcement laboratory with existing technical equipment and staff, the processing of the sequencing data requires additional resources and the appropriate bioinformatic expertise.

Genome Editing to Achieve the Crop Ideotype in Tomato.

For centuries, combining useful traits into a single tomato plant has been done by selective crossbreeding that resulted in hundreds of extant modern cultivars. However, crossbreeding is a labor-intensive process that requires between 5 and 7 years to develop a new variety. More recently, genome editing with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been established as an efficient method to accelerate the breeding process by introducing targeted modifications to plant genomes via generation of targeted double-strand breaks (DSBs). CRISPR/Cas9 has been used to generate a variety of specific changes ranging from gene knockouts to gene replacements, and can also be easily multiplexed to modify several targets simultaneously. Given that (1) generating knockout mutations only requires a DSB that is frequently repaired by the error-prone nonhomologous end joining (NHEJ) pathway resulting in gene function inactivation, and (2) the genetic basis of many useful agronomic traits consists of loss of gene function, multiple traits can be created in a plant in one generation by simultaneously introducing DSBs into multiple genes of interest. On the other hand, more precise modifications, such as allele replacement, can be achieved by gene targeting-a less efficient process in which an external template is used to repair the DSB by homologous recombination (HR). These technical breakthroughs allow the design and customization of plant traits to achieve the ideal plant type ("ideotype"). Here, we describe protocols to assemble CRISPR/Cas9 constructs for both single and multiplex gene knockouts as well as gene targeting and to generate and identify genome-edited tomato plants via Agrobacterium-mediated transformation in tissue culture.

DNA-free genome editing

<p indent=0mm>CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems have been extensively used for gene editing of many plant species, including model plants such as <italic>Arabidopsis</italic> and rice, and major crops such as maize and wheat, as well as woody plants like poplar, bamboo, apple and citrus, etc. This technique has not only significantly promoted basic research, but has also facilitated the agronomic traits improvement of economically important crops or trees. Typically, the CRISPR/Cas system required its integration and expression of exogenous DNA in the genome of host plants, which lead to the cleavage of site-specific DNA double-strand breaks, and introduced the genome modifications during the DNA repair process. However, the integration of CRISPR/Cas elements brings several concerns for commercialization and basic research. First, the inserted CRISPR/Cas cassette is constitutively expressed in plant cells, and may increase the chance of off-target changes, as well as make it difficult to evaluate the stability and penetrance of the initially observed phenotypes. Second, plants with a foreign gene integrated into its genome were regarded as classic genetically modified organisms, which may potentially lead to the escape of the gene into the environment, leading to legislation concerns and strict government policy regulation about such organisms. Therefore, one of the main challenges in using the CRISPR/Cas system in commercial agriculture is to improve the plant’s agronomic traits by genome editing, meanwhile obtaining transgene-free mutant plants with stable transmissible properties. DNA-free genome editing technology in plants is new and began in 2015, and since then great advances have been made on producing transgenic-free genome-edited plants. The exogenous CRISPR/Cas cassette and other transgenes could be eliminated by multiple approaches after the target gene has been edited. For example, the CRISPR/Cas expression cassette can be removed through genetic segregation for plants that are reproduced sexually, and the end products are CRISPR-edited but transgene-free. Transgene-free gene-edited plants may also be generated by non-transgenic approaches, such as transient expression of mRNA encoding Cas nuclease and guide RNA without DNA integration, preassembled ribonucleoprotein and sgRNA complex transfection, fluorescence marker-assisted transgene cassette selection and elimination, virus-mediated and nano-material mediated genome editing technologies. By using these methods, people can obtain the genome-edited end plant product with increased agronomic traits, while no additional foreign DNA fragments have been introduced into the plant genome. Until now, at least 14 transgene-free and genome-edited plant species have been generated using this technology. In this review, we have highlighted these achievements, including methods, applications and the possibilities of their commercialization in plant breeding. We also discuss the current problems in using this technology, such as very low efficiency, limited gene modification types and labor-consuming aspects. With the development of new plant cell transformation methods and the applications of new typical CRISPR/Cas systems, the potential strategies for improving this revolutionary technology in the future are also discussed.

Potato Virus X Vector-Mediated DNA-Free Genome Editing in Plants.

Genome editing technology is important for plant science and crop breeding. Genome-edited plants prepared using general CRISPR-Cas9 methods usually contain foreign DNA, which is problematic for the production of genome-edited transgene-free plants for vegetative propagation or highly heterozygous hybrid cultivars. Here, we describe a method for highly efficient targeted mutagenesis in Nicotiana benthamiana through the expression of Cas9 and single-guide (sg)RNA using a potato virus X (PVX) vector. Following Agrobacterium-mediated introduction of virus vector cDNA, >60% of shoots regenerated without antibiotic selection carried targeted mutations, while ≤18% of shoots contained T-DNA. The PVX vector was also used to express a base editor consisting of modified Cas9 fused with cytidine deaminase to introduce targeted nucleotide substitution in regenerated shoots. We also report exogenous DNA-free genome editing by mechanical inoculation of virions comprising the PVX vector expressing Cas9. This simple and efficient virus vector-mediated delivery of CRISPR-Cas9 could facilitate transgene-free gene editing in plants.

Fertility restoration of Chinese wild rice-type cytoplasmic male sterility by CRISPR/Cas9-mediated genome editing of nuclear-encoded RETROGRADE-REGULATED MALE STERILITY.

Cytoplasmic male sterility (CMS) is a trait that produces nonfunctional pollen caused by the interaction between mitochondrial and nuclear genes. In Chinese-wild (CW) type CMS, CWA, in rice (Oryza sativa L.), its mitochondria enhance the expression of the nuclear gene RETROGRADE-REGULATED MALE STERILITY (RMS), which causes pollen abortion. Fertility is recovered when its expression decreases in a restorer line, CWR. The expression of RMS is controlled by the single nucleotide polymorphism (SNP) located in the promoter region 2,286 bp upstream of the start codon of RMS. However, another gene, PPR2, which encodes pentatricopeptide repeat-domain containing protein, is predicted in the reverse strand of this region and a premature stop codon is created in CWR by the SNP. To prove RMS is directly involved in restoring fertility of CW-CMS, we introduced mutations into RMS and PPR2 using CRISPR/Cas9. Fertility was recovered in the genome-edited CMS plants with reduced expression of RMS and unaltered expression of PPR2, when the mutation was introduced in the promoter regions of RMS within or outside the coding sequence (CDS) of PPR2. Fertility restoration was not obtained when the mutation was introduced within the CDS of RMS. Our results demonstrated that PPR2 is not responsible for fertility restoration, and fertility was recovered by reduced expression of RMS, providing us with a new artificial fertility restorer line for agronomical use.

High frequency direct shoot regeneration from Kazakh commercial potato cultivars

Potato (Solanum tuberosum L.) is the third most economically important crop in the world and has a high nutritional value. In this study, the in vitro culture response of four widely grown in Kazakhstan potato cultivars, Astanalyk, Monument Kunaev, Tokhtar, and Aksor, was investigated using stem and leaf explants. Published protocols were evaluated and optimized to develop a more efficient protocol for the regeneration of plants from local potato cultivars in tissue culture, which is a prerequisite to facilitate potato genome modification. The explants were cultured on solid Murashige and Skoog medium supplemented with different concentrations and combinations of zeatin, 6-benzylaminopurine (BAP), gibberellic acid (GA3), 1-naphthaleneacetic acid (NAA) and indole-3-acetic acid (IAA). The maximum regeneration was induced from the stem internodal explants. A significant effect of the explant source on direct regeneration was confirmed with statistical analysis. The number of shoots obtained from the internode was 10.0 from cv. Aksor followed by cvs. Tokhtar and Astanalyk. The medium DRM-VIII with 1 mg/l zeatin, 0.1 mg/l IAA and 7.0 mg/l GA3 was considered the best for direct shoot regeneration and multiple shoot formation from all cultivars. To conclude, we outline a protocol for direct plant regeneration from four potato cultivars. Our findings suggest commercial cultivars Astanalyk and Aksor are good candidates for developing the genome-edited plants through direct shoot regeneration.

Protocol for Agrobacterium-mediated transformation of tall fescue and future perspective on the application of genome editing.

Tall fescue (Festuca arundinacea Schreb.) is a major cool-season perennial grass grown for forage and turf. We have obtained transgenic tall fescue by Agrobacterium-mediated transformation to improve agronomically important traits. In our protocol, we use embryogenic calli derived from not only mature seeds but also shoot tips. Although tall fescue cultivars consist of various genotypes with different genetic variation, we can produce transgenic plants at any time with calli induced from shoot tips of in vitro-maintained responsive genotypes. When the hygromycin phosphotransferase gene is used as a selectable marker, transformants are selected by incubation with 100 mg l-1 hygromycin in both selection and regeneration media. Since tall fescue is an anemophilous species, the cultivation of transgenic plants poses the risk of transgenic pollen flow. Recently, it has been reported that genome-edited plants without the integration of foreign DNA fragments can be produced by an Agrobacterium-mediated transient gene expression system. We hope that our protocol will contribute to production of transgene-free genome-edited tall fescue.

Progress and Challenges in the Improvement of Ornamental Plants by Genome Editing.

Biotechnological approaches have been used to modify the floral color, size, and fragrance of ornamental plants, as well as to increase disease resistance and vase life. Together with the advancement of whole genome sequencing technologies, new plant breeding techniques have rapidly emerged in recent years. Compared to the early versions of gene editing tools, such as meganucleases (MNs), zinc fingers (ZFNs), and transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeat (CRISPR) is capable of altering a genome more efficiently and with higher accuracy. Most recently, new CRISPR systems, including base editors and prime editors, confer reduced off-target activity with improved DNA specificity and an expanded targeting scope. However, there are still controversial issues worldwide for the recognition of genome-edited plants, including whether genome-edited plants are genetically modified organisms and require a safety evaluation process. In the current review, we briefly summarize the current progress in gene editing systems and also introduce successful/representative cases of the CRISPR system application for the improvement of ornamental plants with desirable traits. Furthermore, potential challenges and future prospects in the use of genome-editing tools for ornamental plants are also discussed.